Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is 'Cluster of Differentiation 44' (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as 'reservoirs' for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a 'Trojan Horse'. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

The Influence of pH on the Scleroglucan and Scleroglucan/Borax Systems.

The effects that an increase of environmental pH has on the triple helix of scleroglucan (Sclg) and on the Sclg/borax hydrogel are reported. Rheological experiments show that the hydrogel is less sensitive to pH increase than Sclg alone, while at pH = 14 a dramatic viscosity decrease takes place for both systems. This effect is evidenced also by the reduced water uptake and anisotropic elongation detected, at pH = 14, by the swelling behaviour of tablets prepared with the Sclg/borax system. On the opposite, a different behaviour was observed with guar gum and locust bean gum tablets, tested as reference polysaccharides. The effect of pH on the structure of Sclg and Sclg/borax was investigated also by means of spectroscopic approaches based on the interaction between Congo red (CR) and the Sclg triple helix. Obtained results indicated that the CR absorbance maximum is shifted as a function of pH and by the presence of borax. Principal component analysis allowed very precise identification of the pH value at which the Sclg helix collapses. Molecular dynamics simulations of the Sclg/borax-CR complex indicated that, at physiological pH, only a few ordered configurations are populated, according to the induced circular dichroism (CD) spectrum evidence.

One-step formation and sterilization of gellan and hyaluronan nanohydrogels using autoclave.

The sterilization of nanoparticles for biomedical applications is one of the challenges that must be faced in the development of nanoparticulate systems. Usually, autoclave sterilization cannot be applied because of stability concerns when polymeric nanoparticles are involved. This paper describes an innovative method which allows to obtain, using a single step autoclave procedure, the preparation and, at the same time, the sterilization of self-assembling nanohydrogels (NHs) obtained with cholesterol-derivatized gellan and hyaluronic acid. Moreover, by using this approach, NHs, while formed in the autoclave, can be easily loaded with drugs. The obtained NHs dispersion can be lyophilized in the presence of a cryoprotectant, leading to the original NHs after re-dispersion in water.

Calcium alginate/dextran methacrylate IPN beads as protecting carriers for protein delivery.

In the present study, mechanical and protein delivery properties of a system based on the interpenetration of calcium-alginate (Ca-Alg) and dextran-methacrylate (Dex-MA) networks are shown. Interpenetrated hydrogels beads were prepared by means of the alginate chains crosslinking with calcium ions, followed by the exposure to UV light that allows the Dex-MA network formation. Optical microscope analysis showed an average diameter of the IPN beads (Ca-Alg/Dex-MA) of 2 mm. This dimension was smaller than that of Ca-Alg beads because of the Dex-MA presence. Moreover, the strength of the IPN beads, and of their corresponding hydrogels, was influenced by the Dex-MA concentration and the crosslinking time. Model proteins (BSA and HRP) were successfully entrapped into the beads and released at a controlled rate, modulated by changing the Dex-MA concentration. The enzymatic activity of HRP released from the beads was maintained. These novel IPN beads have great potential as protein delivery system.

Evaluation of rheological properties and swelling behaviour of sonicated scleroglucan samples.

Scleroglucan is a natural polysaccharide that has been proposed for various applications. However there is no investigation on its property variations when the molecular weight of this polymer is reduced. Scleroglucan was sonicated at two different polymer concentrations for different periods of time and the effect of sonication was investigated with respect to molecular weight variations and rheological properties. Molar mass, estimated by viscometric measurements, was drastically reduced already after a sonication for a few min. Sonicated samples were used for the preparation of gels in the presence of borate ions. The effect of borax on the new samples was investigated by recording the mechanical spectra and the flow curves. A comparison with the system prepared with the dialysed polymer was also carried out. The anisotropic elongation, observed with tablets of scleroglucan and borax, was remarkably reduced when the sonicated samples were used for the preparation of the gels.

Therapeutic effects of dexamethasone-loaded hyaluronan nanogels in the experimental cholestasis.

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na+/H+ exchanger isoform 1 (NHE1) and Cl-/HCO3- exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids' systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies.

Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting.

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.

A new vesicle-loaded hydrogel system suitable for topical applications: preparation and characterization.

PURPOSE. Aim of this research was to prepare and study drug release from a new formulation consisting of non ionic surfactant vesicular structures, niosomes (NSVs), loaded with model molecules calcein (CALC), nile red (NR), ibuprofen (IBU) or caffeine (CAFF), and embedded in a hydrogel matrix. METHODS. The system locust bean gum/xanthan (1:1), prepared at 60 °C, was used to entrap the vesicles (Tween 20/cholesterol 1:1), loaded with guest molecules and the release profiles were detected at 32 °C. The hydrogel systems were characterized by means of scanning electron microscopy; niosomes were characterized by means of size and -potential measurements. RESULTS. Size measurements showed that a slight increase in vesicle dimensions occurs after inclusion of CALC or CAFF (hydrophilic molecules) in the vesicular structures. -potential measurements showed that the inclusion of these molecules did not significantly modify the surface charge of empty vesicles. This was probably related to an almost negligible drug adsorption on the vesicle surface. The release from the niosomes-gel systems of two probes (CALC and NR) showed that the diffusion of CALC through the gel was not affected by the niosome entrapment while for NR, the presence of vesicles was crucial. The release profiles from niosomes-gel systems and from the hydrogel alone of model drugs, CAFF and IBU, showed an appreciable difference between the two drugs: the more hydrophilic CAFF was released much faster than IBU. In all release studies turbidity, dimension and -potential analyses indicated that the loaded niosomes were released by the hydrogel matrix without being damaged. CONCLUSIONS. The reported in vitro experiments show the capability of the novel formulation to combine the qualities of both chosen single systems, i.e. the niosomes and the polymeric network. The hydrogel shows a protective effect on vesicle integrity and leads to a slow release of the loaded model molecules from the polysaccharidic system. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Hyaluronan-Cholesterol Nanogels for the Enhancement of the Ocular Delivery of Therapeutics.

The anatomy and physiology of the eye strongly limit the bioavailability of locally administered drugs. The entrapment of therapeutics into nanocarriers represents an effective strategy for the topical treatment of several ocular disorders, as they may protect the embedded molecules, enabling drug residence on the ocular surface and/or its penetration into different ocular compartments. The present work shows the activity of hyaluronan-cholesterol nanogels (NHs) as ocular permeation enhancers. Thanks to their bioadhesive properties, NHs firmly interact with the superficial corneal epithelium, without penetrating the stroma, thus modifying the transcorneal penetration of loaded therapeutics. Ex vivo transcorneal permeation experiments show that the permeation of hydrophilic drugs (i.e., tobramycin and diclofenac sodium salt), loaded in NHs, is significantly enhanced when compared to the free drug solutions. On the other side, the permeation of hydrophobic drugs (i.e., dexamethasone and piroxicam) is strongly dependent on the water solubility of the entrapped molecules. The obtained results suggest that NHs formulations can improve the ocular bioavailability of the instilled drugs by increasing their preocular retention time (hydrophobic drugs) or facilitating their permeation (hydrophilic drugs), thus opening the route for the application of HA-based NHs in the treatment of both anterior and posterior eye segment diseases.

Preparation and characterization of novel gellan gum hydrogels suitable for modified drug release.

Innovative hydrogels obtained by physical and chemical crosslinking of deacylated Gellan gum have been characterized in terms of water uptake, rheological properties and compressibility, and the behaviour of the tested materials, according to the type of the obtained network, is thoroughly discussed. The release from the various gels of loaded model molecules of different steric hindrance was also investigated and the trend of the release profiles has been related to the structures proposed for the physical and the chemical hydrogel.

Structural characterization of calcium alginate matrices by means of mechanical and release tests.

In this paper we have concentrated on the characterization of calcium alginate hydrogels loaded with a model drug (myoglobin) by means of a mechanical approach; in addition, release tests of myoglobin from alginate hydrogels were performed. At a fixed temperature, relaxation tests (mechanical study) were carried out on matrices constituted by different polymer concentrations. The interpretation of the relaxation behavior of the different matrices was conducted using the generalized Maxwell model; as a result of this investigation it was possible to conclude that for polymer concentrations greater than 0.5 g/ 100 mL the matrices behaved as solid materials. In addition, it was observed that the mechanical properties of the matrices increased with polymer concentration. With regard to the release tests, the diffusion coefficient of myoglobin in the matrix in relation to polymer concentrations was determined. The mechanical and release data where then analyzed by Flory's theory and by a modified free-volume theory, respectively, to estimate the network mesh size xi. The comparison between the mesh sizes obtained by the two approaches showed a satisfactory agreement for polymer concentrations greater than 0.5 g/100 mL. It should be noted that the approach proposed here to determine the polymeric network meshes is absolutely general and can be advantageously applied to the characterization of other similar polymeric systems.

Intracellular Delivery of Natural Antioxidants via Hyaluronan Nanohydrogels.

Natural antioxidants, such as astaxanthin (AX), resveratrol (RV) and curcumin (CU), are bioactive molecules that show a number of therapeutic effects. However, their applications are remarkably limited by their poor water solubility, physico-chemical instability and low bioavailability. In the present work, it is shown that self-assembled hyaluronan (HA)-based nanohydrogels (NHs) are taken up by endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs), preferentially accumulating in the perinuclear area of oxidatively stressed HUVECs, as evidenced by flow cytometry and confocal microscopy analyses. Furthermore, NHs are able to physically entrap and to significantly enhance the apparent water solubility of AX, RV and CU in aqueous media. AX/NHs, RV/NHs and CU/NHs systems showed good hydrodynamic diameters (287, 214 and 267 nm, respectively), suitable ζ-potential values (-45, -43 and -37 mV, respectively) and the capability to neutralise reactive oxygen species (ROS) in tube. AX/NHs system was also able to neutralise ROS in vitro and did not show any toxicity against HUVECs. This research suggests that HA-based NHs can represent a kind of nano-carrier suitable for the intracellular delivery of antioxidant agents, for the treatment of oxidative stress in endothelial cells.

Theoretical and experimental study on a self-assembling polysaccharide forming nanochannels: static and dynamic effects induced by a soft confinement.

It is well-known that the polysaccharide scleroglucan (Sclg) exhibits a triple-helix conformation (triplex) and it is able to form hydrogels in water solution. Furthermore, these hydrogels are influenced by the presence of borax, in terms of rheological and drug release properties. In previous works, we showed that the presence of borax stabilizes the intertriplex interactions and that the property variations, induced by borax, can be fully explained, considering that the Sclg triplexes can form nanochannel-like structures. In this paper, the stability of these aggregates has been experimentally studied by means of atomic force microscopy (AFM) and theoretically investigated by means of molecular dynamics (MD) simulations. The simulations indicate that the borax stabilizes nanochannel-like structures when seven triplexes are considered. The simultaneous presence of different Sclg triplexes in a narrow space strongly influences the properties of confined water molecules in a way similar, in many aspects, to that of water molecules located in the inner part of well-defined nanochannels (e.g., diffusion inside carbon nanotubes). As a consequence, also the conformational properties of flanking regions of Sclg triplexes are influenced. Furthermore, differential scanning calorimetry (DSC) data show that the well-known conformational transition occurring at 280 K for Sclg does not take place in the presence of borax. The MD simulations suggest that such lack of transition is a direct consequence of the presence of borax. The role of Na+ counterions in the hydrogel structure is also investigated.

In situ cross-linkable novel alginate-dextran methacrylate IPN hydrogels for biomedical applications: mechanical characterization and drug delivery properties.

In situ polymerizable hydrogels are extensively investigated to implement new biomedical and pharmaceutical approaches. In the present paper a novel polysaccharidic matrix based on calcium alginate (Ca(II)-Alg) hydrogel and dextran methacrylate derivative (Dex-MA), showing potential applicability in the field of pharmaceutics is described. The semi-interpenetrating polymer system (semi-IPN) obtained by a dispersion of Dex-MA chains into a Ca(II) hydrogel leads to a hydrogel with rheological properties quite different from those of Ca(II)-Alg, allowing to inject the semi-IPN easily through an hypodermic needle. The UV curing of the semi-IPN, by cross-linking of the methacrylate moieties, leads to an IPN strong hydrogel that can be used for a modulated delivery of bioactive molecules. In the present paper, rheological and mechanical behaviors of the semi-IPN and of the IPN are discussed. The release of model molecules, including a protein, are also presented to show the suitability of the novel system as a drug delivery system.

Recent advances and perspectives on coated alginate microspheres for modified drug delivery.

BACKGROUND: Alginate microspheres represent a useful tool for modified drug delivery. Their preparation is quite easy and is usually based on the gelling properties of the polysaccharide in the presence of divalent ions; nevertheless, microparticles prepared only with calcium alginate show several problems, mainly related to the mechanical stability and to the release that, in most cases, is too fast. To overcome such inconveniences, polymer-coated alginate microspheres and/or appropriately interpenetrating polymer network (semi-IPNs and IPNs) structures formed with alginate and other macromolecules were developed. OBJECTIVE: This article reports a synthetic overview on the most recent searches carried out on coated alginate microspheres. METHODS: After a section focused on the microsphere preparation, this article is divided into several main topics related to the specific polymer that was used as a coating material to provide a rationale in reporting literature data. In the last section, the advantages and disadvantages of the various approaches are discussed and the authors' opinion on perspectives for further studies and novel applications of coated alginate microspheres are reported. CONCLUSION: Ca(2+)-alginate microparticles could experience a new era if scientists will increase their efforts in developing microparticles with smart properties.

Long-Circulating Hyaluronan-Based Nanohydrogels as Carriers of Hydrophobic Drugs.

Nanohydrogels based on natural polymers, such as polysaccharides, are gaining interest as vehicles for therapeutic agents, as they can modify the pharmacokinetics and pharmacodynamics of the carried drugs. In this work, hyaluronan-riboflavin nanohydrogels were tested in vivo in healthy rats highlighting their lack of toxicity, even at high doses, and their different biodistribution with respect to that of native hyaluronan. They were also exploited as carriers of a hydrophobic model drug, the anti-inflammatory piroxicam, that was physically embedded within the nanohydrogels by an autoclave treatment. The nanoformulation was tested by intravenous administration showing an improvement of the pharmacokinetic parameters of the molecule. The obtained results indicate that hyaluronan-based self-assembled nanohydrogels are suitable systems for low-soluble drug administration, by increasing the dose as well as the circulation time of poorly available therapeutic agents.

Hyaluronan-Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes.

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.

Two galactomannans and scleroglucan as matrices for drug delivery: preparation and release studies.

Two galactomannans, Guar gum and Locust bean gum, have been used as matrices for tablets to study the release of model molecules. As a comparison, matrices obtained with another polysaccharide, Scleroglucan, have been tested. Despite the different conformations that the polymers assume in aqueous solution (flexible coils for Guar gum and Locust bean gum; triple helix for Scleroglucan), when prepared as tablets, they show (in distilled water and at 37 degrees C) very similar release profiles of guest molecules (i.e. theophylline, vitamin B12 and myoglobin) of different steric hindrance. Furthermore, the polymers were chemically crosslinked with glutaraldehyde to obtain a network suitable as a matrix for modified drug release. The delivery of the model molecules from the Guar gum and Locust bean gum gels, and from tablets prepared from the freeze-dried hydrogels of the three polymers was evaluated, and a comparison with the tablets prepared with the not-crosslinked polymers was carried out. Experimental data showed how the presence in the matrix of a well-defined network, by introducing a spacer among the macromolecular chains, always increased the rate of delivery of the tested molecules in comparison to the release profiles obtained when no crosslinker was present. Release data from the tablets were analyzed according to a mathematical model able to determine the relative importance of drug dissolution and drug diffusion on the overall release kinetics. Good agreement was found between the simulated and the experimental data.

"Click" hyaluronan based nanohydrogels as multifunctionalizable carriers for hydrophobic drugs.

Highly hydrophilic and biocompatible nanocarriers based on polysaccharide hydrogels (nanohydrogels, NHs) were shown to be promising systems for drug delivery applications. Following the idea of these emerging drug carriers, the aim of the present work was to develop self-assembled hydrogel nanoparticles based on amphiphilic derivatives of hyaluronic acid (HA) and riboflavin (Rfv), synthesized by "click" Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction. The obtained amphiphilic product (HA-c-Rfv) was able to form nanohydrogels in aqueous environments, in particular by applying an innovative autoclave-based method. HA of different molecular weights (Mw) and degrees of substitution (DS) were prepared and the effect of these parameters on the NHs formation was assessed. The derivative HA220-c-Rfv 40/40 was chosen as the most interesting system, capable to form NHs in the range of 150-200nm and with a negative ζ-potential. NHs were very stable in water solutions and, by adding dextrose as cryoprotectant, it was also possible to freeze-dry the NHs formulation. The developed system is proposed for the delivery of hydrophobic drugs; for this purpose, dexamethasone, piroxicam and paclitaxel were used as model drugs; these molecules were loaded into NHs with high efficiency by film-hydration technique. Furthermore, a HA-c-Rfv derivative bearing an excess of propargylic portions was capable to react with other N3-derivatized molecules, opening the route to a wide spectrum of functionalization opportunities: in this direction, PEG-N3 has been tested as a model molecule for the preparation of PEGylated NHs.

Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase.

Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan-riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA-CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121°C for 20min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Young's modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm-associated infections.